Fermentative Production Of Fine Chemicals

Abstract

AbstractThe invention relates to a process the production of at least one microbial metabolite having at least 3 carbon atoms or at least 2 carbon atoms and at least 1 nitrogen atom by means of sugar-based microbial fermentation, comprising:a) the preparation of a sugar-containing liquid medium with a monosaccharide content of more than 20% by weight from a starch feedstock, the sugar-containing liquid medium also comprising the non-starchy solid constituents of the starch feedstock;b) the fermentation of the sugar-containing liquid medium for the production of the metabolite(s); andc) elimination or isolation of at least one metabolite from the fermentation liquor,which comprises culturing, in the sugar-containing liquid medium, a microorganism strain which produces the desired metabolite(s), which liquid medium is obtained by:a1) milling the starch feedstock; anda2) liquefying the millbase in an aqueous liquid in the presence of at least one starch-liquefying enzyme, followed by saccharification using at least one saccharifying enzyme, where at least some of the millbase is liquefied by continuous or batchwise addition to the aqueous liquid.

Information

Applicants

Specification

Fermentative production of fine chemicals
The present invention relates to the fermentative production of fine chemicals by grinding, liquefying and saccharifying starch feedstocks and to the use of the resulting sugar solution as fermentation medium.
Fermentative processes for the production of fine chemicals such as, for example, amino acids, vitamins and carotenoids by means of microorganisms are generally known. Depending on the various process conditions, they exploit different carbon feedstocks. They extend from pure sucrose via beet and sugarcane molasses to what are known as high-test molasses (inverted sugarcane molasses) to glucose from starch hydrolyzates. Moreover, acetic acid and ethanol are mentioned as cosubstrates which can be employed on an industrial scale for the biotechnological production of L-lysine (Pfefferle et al., Biotechnogical Manufacture of Lysine, Advances in Biochemical Engineering/Biotechnology, Vol. 79 (2003), 59-112).
Based on the abovementioned carbon feedstocks, various methods and procedures for the sugar-based, fermentative production of fine chemicals are established. Taking L-lysine as an example, these are described for example by Pfefferle et al. (loc. cit.) with regard to strain development, process development and industrial production.
An important carbon feedstock for the microorganism-mediated fermentative production of fine chemicals is starch. The latter must first be liquefied and saccharified in preceding reaction steps before it can be exploited as carbon feedstock in a fermentation. To this end, the starch is usually obtained in pre-purified form from a natural starch feedstock such as potatoes, cassava, cereals, for example wheat, corn, barley, rye or rice, and subsequently enzymaticaliy liquefied and saccharified, whereafter it is employed in the actual fermentation for producing the fine chemicals.
In addition to the use of such pre-purified starch feedstocks, the use of non-pretreated starch feedstocks for the preparation of carbon feedstocks for the fermentative production of fine chemicals has also been described. Typically, the starch feedstocks are initially comminuted by grinding. The millbase is then subjected to liquefaction and saccharification. Since this millbase naturally comprises, besides starch, a series of nonstarchy constituents which adversely affect the fermentation, these constituents are usually removed prior to fermentation. The removal can be effected either directly after grinding (WO 02/277252; JP 2001-072701; JP 56-169594; CN1218111), after liquefaction (WO 02/277252; CN 1173541) or subsequently to saccharification (CN 1266102; Beukema et al.: Production of fermentation syrups by enzymatic hydrolysis of potatoes; potato saccharification to give culture medium (Conference Abstract), Symp. Biotechnol. Res. Neth. (1983), 6; NL8302229). However, all variants involve the use of a substantially pure starch hydrolyzate in the fermentation.

More recent techniques deal in particular with improved methods which are intended to make possible a purification, for example of liquefied and saccharified starch solutions (JP 57159500) and of fermentation media from renewable resources (EP 1205557) prior to fermentation.
Unprocessed starch feedstocks, in contrast, are known to be employed on a large scale in the fermentative production of bioethanol. Here, the method known as "dry milling", liquefaction and saccharification of starch feedstocks is established on a large industrial scale. Suitable process descriptions can be found for example in "The Alcohol Textbook - A reference for the beverage, fuel and industrial alcohol industries", Jaques et al. (ed), Nottingham Univ. Press 1995, ISBN 1-8977676-735 and in McAloon et al., "Determining the cost of producing ethanol from corn starch and lignocellulosic feedstocks" ,NREL/TP-580-28893, National Renewable Energy Laboratory, October 2000.
in the first step of the dry-milling method, whole cereal kernels, preferably corn, wheat, barley, millet and rye, are ground finely. In contrast to what is known as the wet-milling method, no additional liquid is added. The purpose of grinding the material into fine constituents is to make the starch present in the kernels accessible to the effect of water and enzymes in the subsequent liquefaction and saccharification.
Since in the fermentative production of bioethanol the product of value is obtained by distillation, the use of starch feedstocks from the dry-milling process in non-pre-purified form does not constitute a particular problem. However, when using a dry-milling method for the production of fine chemicals, the solids stream introduced into the fermentation via the sugar solution is problematic since it not only may have an adverse effect on the fermentation, but also makes the subsequent processing substantially more difficult.
Thus, the oxygen supply for the microorganisms employed is a limiting factor in many fermentations, in particular when the former have demanding oxygen requirements. In general, little is known about the effect of high solids concentrations on the transition of oxygen from the gas phase into the liquid phase, and thus on the oxygen transfer rate. On the other hand, it is known that a viscosity which increases with increasing solids concentrations leads to a reduced oxygen transfer rate. If, moreover, surface-active substances are introduced into the fermentation medium together with the solids, they affect the tendency of the gas bubbles to coagulate. The resulting bubble size, in turn, has a substantial effect on oxygen transfer (Mersmann, A. et al.: Selection and Design of Aerobic Bioreactors, Chem. Eng. Technol. 13 (1990), 357-370).
As the result of the introduction of solids, a critical viscosity value of the media used can be reached as early as during the preparation of the starch-containing suspension since, for example, a suspension with more than 30% by weight of ground corn in water can no longer be mixed homogeneously (Industrial Enzymology, 2nd ed.,

T.Godfrey, S. West, 1996). This limits the glucose concentration in conventional procedures. As a result, it is disadvantageous for process economical reasons to use solutions with a lower concentration since this results in a disproportionate dilution of the fermentation liquor. This causes the achievable final concentration of the target products to drop, which results in additional costs when these are isolated, and the space-time yield decreases, which, given an equal production quantity, leads to a higher volume requirement, i.e. higher investment costs.
During work-up, the increased solids concentration may result in particular difficulties for the use of specific methods. Thus, for example, when purifying the fermentation liquor by means of ion-exchange chromatography, it must be taken into consideration that the chromatography column employed tends to clogging (i.e. blockage).
Owing to these difficulties, prior-art variants of the dry-milling method are not suitable for providing starch feedstock for the fermentative production of fine chemicals and are therefore without particular economical importance. To date, attempts to apply the dry-milling concept and the advantages which exist in principle in connection with this method, to the industrial-scale production of fine chemicals have only been described using cassava as starch feedstock.
Thus, while JP 2001/275693 describes a method for the fermentative production of amino acids in which peeled cassava tubers which have been ground in the dry state are employed as starch feedstock, it is necessary, to carry out the process, to adjust the particle size of the millbase at < 150 μm. In the filtration step which is employed for this purpose, more than 10% by weight of the millbase employed, including non-starch-containing constituents, are removed before the starch obtained is liquefied/ saccharified and subsequently fermented. Moreover, the method dispenses with the problem of removing non-starch-containing constituents in as far as the fermentation products, for example lysine, are intended to be used as feed additive and the non-starch-containing cassava constituents may thus remain in the product of value.
A similar method is described in JP 2001/309751 for the production of an amino-acid-containing feed additive. Analogously, a purification, or removal of solids, is not
required.-
However,- cassava is relatively problem-free in relation to the dry-milling process in
'comparison with other starch feedstocks. While the starch typically accounts for 80 to
89% .by weight of the dry cassava root (Menezes et al., Fungal celluloses as an aid for
the: saecharification of Cassava, Biotechnology and Bioengineering, Vol. XX, 1978,
John Wiley and Sons, Inc., Table 1, page 558), the starch content (dry matter) in cereal
is comparatively much lower, for example it amounts to approximately 68% by weight
in the case of corn and to approximately 65% by weight in the case of wheat (Jaques et
al., The Alcohol Textbook, ibid.). Accordingly, the glucose solution obtained after
liquefaction and saecharification comprises fewer contaminants, in particular fewer

solids, when employing dry-milled cassava than when employing another dry-milled starch feedstock.
An increased amount of contaminations substantially increases the viscosity of the reaction mixture. Cassava starch, however, is relatively easy to process. While it has a higher viscosity at the swelling temperature in comparison with corn starch, the viscosity, in contrast, drops more rapidly at increasing temperatures than in the case of corn starch (Menezes, TJ.B. de, Saccharification of Cassava for ethyl alcohol production, Process Biochemistry, 1978, page 24, right column). Moreover, the swelling and gelatinization temperatures of cassava starch are lower than those of starch from cereals such as corn, which is why it is more readily accessible to bacterial α-amylase than cereal starch (Menezes, TJ.B. de, loc. cit).
Further advantages of cassava over other starch feedstocks are its low cellulose content and its low phytate content. Cellulose and hemicellulose can be converted into furfurals, in particular under acidic saccharification conditions (Jaques et al., The Alcohol Textbook, ibid.; Menezes, TJ.B. de, ibid.) which, in turn, may have an inhibitory effect on the microorganisms employed in the fermentation. Phytate likewise inhibits the microorganisms employed for the fermentation.
While it is thus possible, from a technical aspect, to process cassava as starch feedstock in a process which corresponds to the dry-milling process, such a cassava-based process is still complex, not optimized and therefore not widely used.
It was thus an object of the present invention to provide an efficient process for the fermentative production of fine chemicals which permits the use of a multiplicity of starch-containing, worldwide locally available plants, for example cereals or potatoes, as starch feedstock. The process was to be distinguished by easy handling of the media used and was to avoid, in particular, complicated pre-purification or main purification steps, such as, for example, the removal of solid non-starch-containing constituents, prior to fermentation. Moreover, it was to allow easy processing of the fermentation mixture. In connection with work carried out by the applicant company, it has been found, surprisingly, that such a process can be carried out in an efficient manner, despite the inherently increased introduction of solids.
The invention thus relates to a process for the production of at least one microbial metabolite having at least 3 carbon atoms, or having at least 2 carbon atoms and at least 1 nitrogen atom by means of sugar-based microbial fermentation, comprising:
a) the preparation of a sugar-containing liquid medium with a monosaccharide content of more than 20% by weight from a starch feedstock, the sugar-containing liquid medium also comprising the non-starch-containing solid constituents of the starch feedstock;

b) the fermentation of the sugar-containing liquid medium for the production of the metabolite(s); and
c) depletion or isolation of at least one metabolite from the fermentation liquor,
which comprises culturing, in the sugar-containing liquid medium, a microorganism strain which produces the desired metabolite(s), which liquid media is obtained by:
a1) milling the starch feedstock; and
a2) liquefying the millbase in an aqueous liquid in the presence of at least one starch-liquefying enzyme, followed by saccharification using at least one saccharifying enzyme, where at least some of the millbase is liquefied by continuous or batchwise addition to the aqueous liquid.
Suitable as starch feedstock are, mainly, dry grains or seeds where the starch amounts to at least 40% by weight and preferably at least 50% by weight in the dried state. They are found in many of the cereal plants which are currently grown on a large scale, such as corn, wheat, oats, barley, rye, rice and various sorghum and millet species, for example sorgo and milo. The starch feedstock is preferably selected from among cereal kernels, especially preferably among corn, rye and wheat kernels. In principle, the process according to the invention can also be carried out with other starch feedstocks such as, for example, potatoes, tapioca or a mixture of various starch-containing fruits or seeds.
The sugars present in the sugar-containing liquid medium are preferably monosaccharides such as hexoses and pentoses, for example glucose, fructose, mannose, galactose, sorbose, xylose, arabinose and ribose, in particular glucose. The amount of monosaccharides other than glucose can vary, depending on the starch feedstock used and the non-starchy constituents present therein; it may be affected by the conduct of the reaction, for example by the decomposition of cellulose constituents by addition of celluiases. The monosaccharides of the sugar-containing liquid medium advantageously comprise glucose in an amount of at least 60% by weight, preferably at least 70% by weight, especially preferably at least 80% by weight, based on the total amount of sugars present in the sugar-containing liquid medium. Usually, the glucose amounts to in the range of from 75 to 99% by weight, in particular from 80 to 97% by weight and specifically from 85 to 95% by weight, based on the total amount of sugars present in the sugar-containing liquid medium.
To prepare the sugar-containing liquid medium, the starch feedstock in question is milled in stepal), with or without addition of liquid, for example water, preferably without addition of liquid. It is also possible to combine dry milling with a subsequent wet-milling step. Apparatuses which are typically employed for dry milling are hammer mills, rotor mills or roller crushers; those which are suitable for wet grinding are paddle

mixers, agitated ball mills, circulation mills, disk mills, annular chamber mills, oscillatory mills or planetary mills. In principle, other mills are also suitable. The amount of liquid required for wet grinding can be determined by the skilled worker in routine experiments. It is usually adjusted in such a way that the dry matter content is in the range of from 10 to 20% by weight.
Grinding brings about a particle size which is suitable for the subsequent process steps. In this context, it has proved advantageous when the millbase obtained in the milling step, in particular the dry milling step, in stepal) has flour particles, i.e. particulate constituents, with a particle size in the range of from 100 to 630 μm in an amount of from 30 to 100% by weight, preferably 40 to 95% by weight and especially preferably 50 to 90% by weight. Preferably, the millbase obtained comprises 50% by weight of flour particles with a particle size of more than 100μm. As a rule, at least 95% by weight of the flour particles obtained have a particle size of less than 2 mm. In this context, the particle size is measured by means of screen analysis using a vibration analyzer. In principle, a small particle size is advantageous for obtaining a high product yield. However, an unduly small particle size may result in problems, in particular problems due to clump formation/agglomeration, when the millbase is slurried during liquefaction or processing, for example during drying the solids after the fermentation step.
Usually, flours are characterized by the extraction rate or by the flour grade, whose correlation with one another is such that the characteristic of the flour grade increases with increasing extraction rate. The extraction rate corresponds to the amount by weight of the flour obtained based on 100 parts by weight of millbase applied. While, during the milling process, pure, ultrafine flour, for example from the interior of the cereal kernel, is initially obtained, the amount of crude fiber and husk content in the flour increases, while the proportion of starch decreases. The extraction rate is therefore also reflected in what is known as the flour grade, which is used as a figure for classifying flours, in particular cereal flours, and which is based on the ash content of the flour (known as ash scale). The flour grade or type number indicates the amount of ash (minerals) in mg which is left behind when 100 g of flour solids are incinerated. In the case of cereal flours, a higher type number means a higher extraction rate since the core of the cereal kernel comprises approximately 0.4% by weight of ash, while the husk comprise approximately 5% by weight of ash. In the case of a lower extraction rate, the cereal flours thus consist predominantly of the comminuted endosperm, i.e. the starch content of the cereal kernels; in the case of a higher extraction rate, the cereal flours also comprise the comminuted, protein-containing aleurone layer of the grains; in the case of coarse mill, they also comprise the constituents of the protein-containing and fat-containing embryo and of the husks, which comprise raw fiber and ash. For the purposes of the invention, flours with a high extraction rate, or a high type number, are preferred in principle. If cereal is employed as starch feedstock, it is preferred that the intact kernels together with their husks are milled and processed.

If appropriate, the starch feedstock will, prior to milling, be comminuted to a size which is suitable for milling, for example when using relatively large materials such as potatoes or cassava. In the case of cereals, this comminution step can be dispensed with, and the intact kernel is employed and milled.
To liquefy the starch present in the millbase, at least some of the millbase, preferably at least 40% by weight, in particular at least 50% by weight and very especially preferably at least 55% by weight, are introduced, in step a2), into the reactor in the course of the liquefaction step, but before the saccharification step. Frequently, the added amount will not exceed 90% by weight, in particular 85% by weight and especially preferably 80% by weight. Preferably, this part of the millbase which is added in the course of the process is supplied to the reactor under conditions as prevail during the liquefaction step. The addition can be effected batchwise, i.e. portionwise, in several portions which preferably in each case do not amount to more than 20% by weight, especially preferably not more than 10% by weight, for example 1 to 20% by weight, in particular 2 to 10% by weight, of the total amount of the millbase to be liquefied, or else continuously. It is essential for the invention that only some of the millbase, preferably not more than 60% by weight, in particular not more than 50% by weight and especially preferably not more than 45% by weight of the millbase are present in the reactor at the beginning of the liquefaction process and that the remainder of the millbase is added during the liquefaction step. The liquefaction can also be carried out continuously, for example in a multi-step reaction cascade.
In accordance with the invention, the liquefaction in step a2) is carried out in the presence of at least one starch-liquefying enzyme which is preferably selected from the α-amylases. Other enzymes which are active and stable under the reaction conditions and which liquefy stable starch can likewise be employed.
The a-amylase (or the starch-liquefying enzyme used) can be introduced first into the reaction vessel or added in the course of step a2). Preferably, some of the a-amylase required in step a2) is added at the beginning of step a2) or is first placed into the reactor. The total amount of a-amylase is usually in the range of from 0.002 to 3.0% by weight, preferably from 0.01 to 1.5% by weight and especially preferably from 0.02 to 0.5% by weight, based on the total amount of starch feedstock employed.
The liquefaction can be carried out above or below the gelatinization temperature. Preferably, the liquefaction in step a2) is carried out at least in part above the gelling temperature of the starch employed (known as the cooking process). As a rule, a temperature in the range of between 70 and 165°C, preferably between 80 and 125°C and especially preferably between 85 and 115°C is chosen, the temperature preferably being at least 5°C and especially preferably at least 10°C above the gelling temperature.
To achieve an optimal a-amylase activity, step a2) is preferably at least in part carried

out at a pH in the weakly acidic range, preferably between 4.0 and 7.0, especially preferably between 5.0 and 6.5, the pH usually being adjusted before or at the beginning of step a2); preferably, this pH is checked during the liquefaction and, if appropriate, readjusted. The pH is preferably adjusted using dilute mineral acids such as H2S04 or H3PO4, or dilute alkali hydroxide solutions such as NaOH or KOH.
In a preferred embodiment, step a2) of the process according to the invention is carried out in such a way that a portion amounting to not more than 60% by weight, preferably not more than 50% by weight and especially preferably not more than 45% by weight, for example 10 to 60% by weight, in particular 15 to 50% by weight, and especially preferably 20 to 45% by weight, based on the total amount of millbase, is initially suspended in an aqueous liquid, for example fresh water, recirculated process water, for example from the fermentation or the processing stages, or in a mixture of these liquids, and the liquefaction is subsequently carried out.
To carry out the method according to the invention, it is possible to preheat the liquid used for generating the suspension to a moderately increased temperature, for example in the range of from 40 to 60°C. However, it is preferred to employ the liquids at room temperature.
Then, the at least one starch-liquefying enzyme, preferably an a-amylase, is added to this suspension. If an a-amylase is used, it is advantageous only to add some of the a-amylase, for example 10 to 70% by weight, in particular 20 to 65% by weight, based on all of the a-amylase employed in step a2). The amount of a-amylase added at this point in time depends on the activity of the a-amylase in question under the reaction conditions with regard to the starch feedstock used and is generally in the range of from 0.0004 to 2.0% by weight, preferably from 0.001 to 1.0% by weight and especially preferably from 0.02 to 0.3% by weight, based on the total amount of the starch feedstock employed. As an alternative, the a-amylase portion can be mixed with the liquid used before the suspension is made.
In this context, the a-amylase portion is preferably added before heating to the temperature used for the liquefaction has started, in particular at room temperature or only moderately increased temperature, for example in the range of from 20 to 30°C.
Advantageously, the amounts of a-amylase and millbase will be selected in such a way that the viscosity during the gelling process is sufficiently reduced in order to make possible effective mixing of the suspension, for example by means of stirring. Preferably, the viscosity of the reaction mixture during gelling amounts to not more than 20 Pas, especially preferably not more than 10 Pas and very especially preferably not more than 5 Pas. As a rule, the viscosity is measured using a Haake viscometer type Roto Visko RV20 with M5 measuring system and MVDIN instrumentation at a temperature of 50°C and a shear rate of 200 s-1.

The suspension thus made is then heated, preferably at a temperature above the gelling temperature of the starch used. As a rule, a temperature in the range of between 70 and 165°C, preferably between 80 and 125°C and especially preferably between 85 and 115°C is chosen, the temperature preferably being at least 5°C and especially preferably at least 10°C above the gelling temperature. While monitoring the viscosity, further portions of the starch feedstock, for example in each case 2 to 20% by weight and in particular from 5 to 10% by weight, based on all of the starch employed, are added gradually to the starch-containing suspension. It is preferred to add the portion of the millbase to be added in the course of the liquefaction step in at least 2, preferably at least 4 and especially preferably at least 6 fractions to the reaction mixture. As an alternative, the portion of the millbase which has not employed for making the suspension can be added continuously during the liquefaction step. During the addition, the temperature should advantageously be kept above the gelling temperature of the starch.
After all of the flour has been added, the reaction mixture is usually held for a certain period of time, for example 30 to 60 minutes or longer, if necessary, at the temperature set above the gelling temperature of the starch, i.e. cooked. Then, the reaction mixture is, as a rule, cooled to a temperature slightly less above the gelling temperature, for example 75 to 90°C, before a further a-amylase portion, preferably the main portion, is added. Depending on the activity under the reaction conditions of the a-amylase used, the amount of a-amylase added at this point in time is preferably 0.002 to 2.0% by weight, especially preferably from 0.01 to 1.0% by weight and very especially preferably from 0.02 to 0.4% by weight, based on the total amount of the starch feedstock employed.
At these temperatures, the granular structure of the starch is destroyed (gelling), making possible the enzymatic degradation of the latter. To fully degrade the starch into dextrins, the reaction mixture is held at the set temperature, or, if appropriate, heated further, until the detection of starch by means of iodine or, if appropriate, another test for detecting starch is negative or at least essentially negative. If appropriate, one or more further a-amylase portions, for example in the range of from 0.001 to 0.5% by weight and preferably from 0.002 to 0.2% by weight, based on the total amount of the starch feedstock employed, may now be added to the reaction mixture.
After the starch liquefaction has ended, the dextrins present in the liquid medium are saccharified, i.e. broken down into glucose, either continuously or batchwise, preferably continuously. The saccharification is carried out either in a specific saccharification tank or directly in the fermenter.
In the first case, the liquefied starch solution is usually chilled or warmed to the temperature optimum of the saccharifying enzyme or slightly below, for example to 50 to 70°C, preferably 60 to 65°C, and subsequently treated with glucoamylase.

If the saccharification is carried out in the fermenter, the liquefied starch solution will, as a rule, be cooled to fermentation temperature, i.e. 32 to 37°C, before it is fed into the fermenter. In this case, the glucoamylase (or the at least one saccharifying enzyme) for the saccharification is added directly to the fermentation liquor. The saccharification of the liquefied starch in accordance with step a2) now takes place in parallel with the metabolization of the sugar by the microorganisms as described in step b).
Prior to addition of the glucoamylase, the pH of the liquid medium is advantageously adjusted to a value in the optimal activity range of the glucoamylase employed, preferably in the range of between 3.5 and 6.0; especially preferably between 4.0 and 5.5 and very especially preferably between 4.0 and 5.0.
In a preferred embodiment, the saccharification is carried out in a specific saccharification tank. To this end, the liquefied starch solution is warmed to a temperature which is optimal for the enzyme, or slightly below, and the pH is adjusted in the above-described manner.
Usually, the glucoamylase is added to the dextrin-containing liquid medium in an amount of from 0.001 to 5.0% by weight, preferably from 0.005 to 3.0% by weight and especially preferably from 0.01 to 1.0% by weight, based on the total amount of the starch feedstock employed. After addition of the glucoamylase, the dextrin-containing suspension is preferably held for a period of, for example 2 to 72 hours or longer, if required, in particular 5 to 48 hours, at the set temperature, the dextrins being saccharified to give monosaccharides. The progress of the saccharification process can be monitored using methods known to the skilled worker, for example HPLC, enzyme assays or glucose test strips. The saccharification is complete when the monosaccharide concentration no longer rises substantially, or indeed drops.
In a preferred embodiment, the discontinuous or continuous addition, preferably the discontinuous and in particular portionwise addition, of the millbase in the presence of the at least one a-amyiase and the at least one glucoamylase in step a2) is carried out in such a way that the viscosity of the liquid medium is not more than 20 Pas, preferably not more than 10 Pas and especially preferably not more than 5 Pas. To aid the control of the viscosity, it has proved advantageous to add at least 25% by weight, preferably at least 35% by weight and especially preferably at least 50% by weight of the total amount of the added millbase at a temperature above the gelatinization temperature of the starch present in the millbase. Moreover, controlling the viscosity can furthermore be influenced by adding the at least one starch-liquefying enzyme, preferably an a-amyiase, and/or the at least one saccharifying enzyme, preferably a glucoamylase, portionwise themselves.
By practicing steps a1) and a2), it is possible to produce the sugar-containing liquid with a monosaccharide content of preferably more than 30% by weight and especially

preferably more than 40% by weight.
Enzymes which can be used for liquefying the starch portion in the millbase are, in principle, all the a-amylases (enzyme class EC 3.2.1.1), in particular a-amylases obtained from Bacillus lichenformis or Bacillus staerothermophilus and specifically those which are used for liquefying materials obtained by dry-milling methods in connection with the production of bioethanol. The a-amylases which are suitable for the liquefaction are also commercially available, for example from Novozymes under the name Termamyl 120 L, type L; or from Genencor under the name Spezyme. A combination of different a-amylases may also be employed for the liquefaction.
Enzymes which can be used for saccharifying dextrins (i.e. oligosaccharides) in the liquefied starch solution are, in principle, all the glucoamylases (enzyme class EC 3.2.1.3), in particular glucoamylases obtained from Aspergilus and specifically those which are used for saccharifying materials obtained by dry-milling methods in connection with the production of bioethanol. The glucoamylases which are suitable for the saccharification are also commercially available, for example from Novozymes under the name Dextrozyme GA; or from Genencor under the name Optidex. A combination of different glucoamylases may also be used.
To stabilize the enzymes employed, the concentration of Ca2+ ions may, if appropriate, be adjusted to an enzyme-specific optimum value, for example using CaCI2. Suitable concentration values can be determined by the skilled worker in routine experiments. If, for example, Termamyl is employed as a-amylase, it is advantageous to adjust the Ca2+ concentration to for example 50 to 100 ppm, preferably 60 to 80 ppm and especially preferably about 70 ppm in the liquid medium.
Since the entire starch feedstock is used for the production of the sugar-containing liquid medium of a), for example in the case of cereals the entire kernel, the non-starchy solid constituents of the starch feedstock are also present. This frequently brings about the introduction of an amount of phytate from the cereal, which amount is not to be overlooked. To avoid the inhibitory effect which thus results, it is advantageous to add, in step a2), at least one phytase to the liquid medium before subjecting the sugar-containing liquid medium to the fermentation step b).
The phytase can be added before, during or after the liquefaction or the saccharification, if it is sufficiently stable to the respective high temperatures.
Any phytases can be employed as long as their activity is in each case not more than marginally affected under the reaction conditions. Phytases used preferably have a heat stability (T50) > 50°C and especially preferably > 60°C.
The amount of phytase is usually from 1 to 10 000 units/kg starch feedstock and in particular 20 to 1000 units/kg starch feedstock.

To increase the overall sugar yield, or to obtain free amino acids, further enzymes, for example pullulanases, cellulases, glucanases, xylanases, glucosidases or proteases, may additionally be added to the reaction mixture during the production of the sugar-containing liquid medium. The addition of these enzymes can have a positive effect on the viscosity, i.e. reduced viscosity (for example by cleaving longer-chain glucans and/or (arabino-)xylanes), and bring about the liberation of metabolizable glucosides and the liberation of (residual) starch. The use of proteases has analogous positive effects, it additionally being possible to liberate amino acids which act as growth factors for the fermentation.
The sugar-containing liquid medium can advantageously be used for the fermentative production of a microbial metabolite having at least 3 carbon atoms or at least 2 carbon atoms and at least 1 nitrogen atom. To this end, the sugar-containing liquid medium produced in step a) is subjected to a fermentation as described in b). In the fermentation, fine chemicals, i.e. compounds having at least 3 carbon atoms and/or at least one nitrogen atom and at least 2 carbon atoms, are produced by the microorganisms. As a rule, the fermentation process can be carried out in the usual manner which is known to the skilled worker.
Hereinbelow, the term fine chemical comprises in particular organic mono-, di- and tricarboxylic acids which preferably have 3 to 10 carbon atoms and which, if appropriate, have one or more, for example 1, 2, 3 or 4, hydroxyl groups attached to them, for example tartaric acid, itaconic acid, succinic acid, fumaric acid, maleic acid, 2,5-furandicarboxylic acid, 3-hydroxypropionic acid, glutaric acid, levulic acid, lactic acid, propionic acid, gluconic acid, aconitic acid and diaminopimelic acid, citric acid; proteinogenic and nonproteinogenic amino acids, for example lysine, glutamate, methionin, phenylalanin, aspartic acid and threonin; purine and pyrimidine bases; nucleosides and nucleotides, for example nicotinamide adenine dinucleotide (NAD) and adenosine-S'-monophosphate (AMP); lipids; saturated and unsaturated fatty acids having preferably 10 to 22 carbon atoms, for example Y-Iin0'enic ac'd, dihomo-y-linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid; diols having preferably 3 to 8 carbon atoms, for example propanediol and butanediol; higher-functionality alcohols having 3 or more, for example 3, 4, 5 or 6, OH groups, for example glycerol, sorbitol, mannitoi, xylitol and arabinitol; longer-chain alcohols having at least 4 carbon atoms, for example 4 to 22 carbon atoms, for example butanol; carbohydrates, for example hyaluronic acid and trehalose; aromatic compounds, for example aromatic amines, vanillin and indigo; vitamins and provitamins, for example ascorbic acid, vitamin B6, vitamin B12 and riboflavin, cofactors and what are known as nutraceutics; proteins, for example enzymes; carotenoides, for example lycopene, (3-carotin, astaxanthin, zeaxanthin and canthaxanthin; ketones having preferably 3 to 10 carbon atoms and, if appropriate, 1 or more hydroxyl groups, for example acetone and acetoin; lactones, for example y-butyrolactone, cyclodextrins, Copolymers, for example polyhydroxyacetate, polyesters, polysaccharides, polyisoprenoids,

polyamides, polyhydroxyalkanoates, for example poly-3-hydroxybutyric acid and copolyesters with other organic hydroxycarboxylic acids such as 3-hydroxyvaleric acid, 4-hydroxybutyric acid and others which are described in Steinbuchel (Ed.), Biopoiymers, 1st Ed., 2003, Wiley-VCH, Weinheim, and the literature cited therein; and precursors and derivatives of the abovementioned compounds. Other compounds which are suitable as fine chemicals are described by Gutcho in Chemicals by Fermentation, Noyes Data Corporation (1973), ISBN: 0818805086.
The term "cofactor* comprises nonproteinaceous compounds which are required for the occurrence of a normal enzyme activity. These compounds can be organic or inorganic; preferably, the cofactor molecules of the invention are organic. Examples of such molecules are NAD and nicotinamide adenine dinucleotide phosphate (NADP); the precursor of these cofactors is niacin.
The term "nutraceutical" comprises food additives which promote health in plants and animals, in particular humans. Examples of such molecules are vitamins, antioxidants and certain lipids, for example polyunsaturated fatty acids.
The process according to the invention is preferably employed for the production of nonvolatile microbial metabolites. For the purposes of the present invention, nonvolatile metabolites are understood as meaning compounds which in general cannot be removed by distillation from the fermentation liquor without undergoing decomposition. As a rule, these compounds have a boiling point which is above the boiling point of water, frequently above 150°C, and in particular above 200°C, at atmospheric pressure.
The microorganisms employed in the fermentation depend in a manner known per se on the fine chemicals in question, as specified in detail hereinbelow. They can be of natural origin or genetically modified. Examples of suitable microorganisms and fermentation processes are those given in Table A hereinbelow:

Preferred embodiments of the process according to the invention relate to the production of amino acids, vitamins, precursors and derivatives thereof, such as, in particular, lysine, methionin, threonin, pantothenic acid and riboflavin, and to the production of polyhydroxyalkanoates of the abovementioned mono-, di- and tricarboxylicacids, specifically succinic acid, lactic acid and propionic acid, the abovementioned diois, specifically propanediol, of the abovementioned ionger-chain alkanols, specifically butanol, of the abovementioned ketones, specifically acetone, and of the abovementioned carbohydrates, specifically trehalose.
In a preferred embodiment, the microorganisms employed in the fermentation are therefore selected from among natural or recombinant microorganisms which overproduce amino acids, vitamins, precursors and/or derivatives thereof or polyhydroxyalkanoates, in particular from among microorganisms which overproduce lysine, methionin, pantothenic acid, riboflavin, polyhydroxyalkanoates, succinic acid, lactic acid, threonin, propionic acid, propanediol, butanol, acetone and trehalose.

In particular, the microorganisms are selected from among the genera Corynebacterium, Bacillus, Ashbya, Escherichia, Aspergillus, Alcaligenes, Actinobacillus, Anaerobiospirillum, Lactobacillus, Propionibacterium and Clostridium, in particular, among strains of Corynebacterium glutamicum, Bacillus subtilis, Ashbya gossypii, Escherichia coli, Aspergillus niger or Alcaligenes latus, Anaerobiospirillum succiniproducens, Actinobacillus succinogenes, Lactobacillus delbruckii, Lactobacillus leichmanni, Propionibacterium arabinosum, Propionibacterium schermanii, Propionibacterium freudenreichii, Clostridium propionicum and Clostridium acetobutlicum.
In a specific preferred embodiment, the metabolite produced by the microorganisms in the fermentation is lysine. To carry out the fermentation, analogous conditions and procedures as have been for other carbon feedstocks, for example in Pfefferle et a!., loc. cit. and US 3,708,395, can be employed. In principle, both a continuous and a discontinuous (batch or fed-batch) mode of operation are suitable, with the fed-batch mode being preferred.
In a further especially preferred embodiment, the metabolite produced by the microorganisms in the fermentation is methionin. To carry out the fermentation, analogous conditions and procedures as have been described for other carbon feedstocks, for example in WO 03/087386 and WO 03/100072, may be employed.
In a further especially preferred embodiment, the metabolite produced by the microorganisms in the fermentation is pantothenic acid. To carry out the fermentation, analogous conditions and procedures as have been described for other carbon feedstocks, for example in WO 01/021772, may be employed.
In a further especially preferred embodiment, the metabolite produced by the microorganisms in the fermentation takes the form of polyhydroxyalkanoates such as poly-3-hydroxybutyrate and copolyesters with other organic hydroxycarboxyiic acids such as 3-hydroxyvaleric acid, 4-hydroxybutyric acid and others which are described in Steinbuchel (loc. cit.), including for example longer-chain hydroxycarboxyiic acids such as 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxytetradecanoic acid, and mixtures of these. To carry out the fermentation, analogous conditions and procedures as have been described for other carbon feedstocks, for example in S.Y. Lee, Plastic Bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria, Tibtech, Vol. 14, (1996), pp. 431-438, may be employed.
In a further especially preferred embodiment, the metabolite produced by the microorganisms in the fermentation is riboflavin. To carry out the fermentation, analogous conditions and procedures as have been described for other carbon feedstocks, for example in WO 01/011052, DE 19840709, WO 98/29539, EP 1186664 and Fujioka, K: New biotechnology for riboflavin (vitamin B2) and character of this riboflavin. Fragrance Journal (2003), 31(3), 44-48, may be employed.

The isolation of fine chemicals from the fermentation liquor in accordance with step c) can be performed as one or more steps. An essential step in this context is the removal of the solid constituents from the fermentation liquor. This can be carried out either before or after isolation of the product of value. Methods conventionally used in the art which also comprise steps for the rough cleaning and the fine purification of the products of value and for formulation are known both for the isolation of products of value and for the removal of solids, i.e. solid-liquid phase separation (for example described in Belter, P.A, Bioseparations: Downstream Processing for Biotechnology, John Wiley & Sons (1988), and Ullmann's Encyclopedia of Industrial Chemistry, 5th ed. on CD-ROM, Wiley-VCH).
To isolate the product of value, a procedure can advantageously be followed in which the solid constituents are first removed from the fermentation liquor, for example by means of centrifugation or filtration, and the product of value is subsequently isolated from the liquid phase, for example by crystallization, precipitation, adsorption or distillation. As an alternative, the product of value can also be isolated directly from the fermentation liquor, for example by using chromatographic methods or extractive methods. A chromatographic method which must be mentioned in particular is ion-exchange chromatography, where the product of value can be isolated selectively on the chromatography column. In this case, the removal of the solids from the fermentation liquor which remains is advantageously carried out for example by decanting, evaporation and/or drying.
Examples of conventional filtration methods are cake filtration and depth filtration (for example described in A. Rushton, A.S. Ward.R.G. Holdich: Solid - Liquid Filtration and Separation Technology, VCH Verlagsgesellschaft, Weinheim 1995, pp. 177 ff., K.J. Ives, in A. Rushton (Ed.): Mathematical Models and Design Methods in Solid-Liquid Separation, NATO ASI series E No. 88, Martinus Nijhoff, Dordrecht 1985, pp. 90 ff.) and cross-flow filtrations, in particular microfiltration for the removal of solids > 0.1 nm (for example described in J. Altmann, S. Ripperger, J. Membrane Sci. 124 (1997) 119-128).
Customary centrifugation methods are described for example in G. Hultsch, H. Wilkesmann, "Filtering Centrifuges," in D.B. Purchas, Solid - Liquid Separation, Upland Press, Croydon 1977, pp. 493-559; and H. Trawinski, Die aquivaiente Klarflacbe von Zentrifugen [The equivalent clarifying area of centrifuges], Chem. Ztg. 83 (1959) 606-612. Various designs such as tube centrifuges, basket centrifuges and, specifically, pusher centrifuges, slip-filter centrifuges and disk separators may be employed.
Conventional extraction methods comprise batchwise or stepwise methods and differential continuous methods with cocurrent flow or countercurrent flow. In this context, the method may involve two or one mobile phase(s). The solubility in both

phases, of the product of value and of the secondary components to be removed, can be influenced, inter alia, by the choice of the solvent, the variation of the counterions and by varying the pH (Treybal, R.E., Mass Transfer Operations, 3rd ed., New York, McGraw-Hill, 1979; Kula, M., Kroner, K.H.. Hustedt, H. and Schutee, H., Technical aspects of extractive enzyme purification, Ann. N.Y. Acad. ScL, 341 (1981); Robinson, R.G., and Cha, D.Y., Controlled pH extraction in the separation of weak acids and bases, Biotech. Progress, 1(1), 18 (1985)).
Customary adsorption methods are described, for example in D.M. Ruthven: Principles of Adsorption and Adsorption Processes, J. Wiley & Sons, New York 1984.; G. Wedler: Adsorption, Verlag Chemie, Weinheim 1970. Solid-bed, moving-bed and fluidized-bed adsorbers can be employed. The adsorption can be carried out batchwise or continuously (K. Hauffe, S.R. Morrison: De Gruyter Studienbuch "Adsorption," De Gruyter, Berlin 1974.; W. Kast: Adsorptionstechnik [Adsorption techniques], VCH Verlagsgesellschaft, Weinheim 1988). in addition to many other adsorbents, activated carbons, ion-exchanger resins, natural or synthetic zeolites and activated aluminas can be employed. Besides, affinity adsorption methods may also be employed (for example described in Arnold, F.H., Blanch, H.W. and Wilke, C.R., Analysis of Affinity separations. Chem. Engr. J., 30, B9 (1985)).
Methods which can be employed in particular for purifying the fine chemicals are, for example, chromatography, precipitation, ultrafiltration, microfiltration, nanofiltration, reverse osmosis, electrophoresis, electrodialysis and isoelectric focusing.
Chromatographic methods can be carried out batchwise or continuously. The continuous chromatography includes, for example, a continuous rotating annular chromatograph (CRAC) (for example described in A.J.P. Martin, Discuss. Farraday Soc. 7 (1949)), a true moving-bed chromatograph (TMBC) (for example described in K. Takeuchi, T. Miyauchi, Y. Uraguchi, J. Chem. Eng. Japan 11 (1978) 216-220.) and a simulated moving-bed chromatograph (SMB) (for example described in D.B. Broughton, Universal Oil Products Co., US 2,985,589, 1961). Solid phases which are employed are, for example, activated aluminas, silica gels, glycol-impregnated diatomaceous earths, dextrans, polymers of sulfonated styrenes, polyacrylamides and polymer-bound proteins (Arnold, F.H., Blanch, H.W. and Wilke, C.R., Analysis of Affinity separations. Chem. Engr. J., 30, B9 (1985); Gibbs, S.J., and Lightfoot, E.N., Scaling up gradient elution chromatography, IEC Fund., 25, 490 (1986); King, C.J., Separation Processes, 2nd ed., New York, McGraw-Hill (1979); Yau, W.W., Kirlland, J.J. and Bly, D.D., Modern Size-Exclusion Liquid Chromatography, Wiley, New York (1979)).
A precipitation may involve a precipitation of either the products of value or the secondary components (J.W. Mullin: Crystallization, 3rd ed., Butterworth-Heinemann, Oxford 1993). The precipitation can be initiated for example by addition of a further solvent, addition of salts and the variation of the temperature. The resulting precipitate

can be separated from the liquor by the above-described conventional methods for separating solids.
Examples of materials which can be employed in microfiltration, ultrafiltration, nanofiltration and reverse osmosis are microporous membranes (A.S. Michaels: "Ultrafiltration," in E.S.Perry (ed.): Progress in Separation and Purification, vol.1, Interscience Publ., New York 1968.), homogeneous membranes (J. Crank, G.S. Park (eds.): Diffusion in Polymers, Academic Press, New York 1968; S.A. Stern: "The Separation of Gases by Selective Permeation," in P. Meares (ed.): Membrane Separation Processes, Elsevier, Amsterdam 1976), asymmetric membranes (R.E. Kesting: Synthetic Polymeric Membranes, A Structural Perspective, Wiley-lnterscience, New York 1985) and electrically charged membranes (F. Helfferich: Ion-Exchange, McGraw-Hill, London 1962), all of which are produced by different methods (R. Zsigmondy, US 1 421 341, 1922; D.B. Pall, US 4 340 479, 1982; S. Loeb, S. Sourirajan, US 3133 132, 1964). Typical materials are cellulose esters, nylon, polyvinyl chloride, acrylonitrile, polypropylene, polycarbonate and ceramics. Thse membranes are employed as a plate module (R.F. Madsen, Hyperfiltration and Ultrafiltration in Plate-and-Frame Systems, Elsevier, Amsterdam 1977), spiral module (US 3 417 870, 1968 (D.T. Bray)), tube bundle or hollow-fiber module (H. Strathmann: "Synthetic Membranes and their Preparation," in M.C. Porter (ed.): Handbook of Industrial Membrane Technology, Noyes Publication, Park Ridge, NJ 1990, pp. 1-60). In addition, the use of liquid membranes is possible (N.N. Li: "Permeation Through Liquid Surfactant Membranes," AlChE J. 17 (1971) 459; S.G. Kimura, S.L Matson, W.J. Ward III: "Industrial Applications of Facilitated Transport," in N.N. Li (ed.): Recent Developments in Separation Science, vol. V, CRC Press, Boca Raton, Florida, 1979, pp. 11-25). The desired product of value can not only be enriched on the feed side and removed via the retentate stream, but also depleted on the feed side and removed via the filtrate/permeate strea.
Electrophoretic methods are described, for example in Rudge, S.R., Ladisch, M.R., Process considerations for scale-up of liquid chromatography and electrophoresis, in Separation Recovery and Purification in Biotechnology, J. Asenjo and J. Hong, eds., ACS Symposium Series, 314, 122 (1986). A large number of variants such as, for example, isoelectric focusing in granulated gel layers, continuous isoelectric focusing with recycling, the "Rotofor" cell, free-flow focusing with recycling and multi-compartmental electrolysis with isoelectric membranes are used. Matrix materials which are employed are, inter alia, cellulose acetate, agarose gels and polyacrylamide gels.
Customary crystallization methods are described, for example, in Janeic, S.J., Grootscholten, P.A., Industrial Crystallization, New York, Academic, 1984; A.W. Bamforth: Industrial Crystallization, Leonard Hill, London 1965; G. Matz: Kristallisation, 2nd ed., Springer Verlag, Berlin 1969; J. Nyvlt: Industrial Crystallization —State of the Art. VCH Verlagsges., Weinheim 1982; S.J. Janeic,

PAM. Grootscholten: Industrial Crystallization, Reidel, Dordecht 1984; O. Sohnel, J. Garside: Precipitation, Butterworth-Heinemann, Oxford, 1992; A.S. Myerson (ed.): Handbook of Industrial Crystallization, Butterworth-Heineman, Boston 1993; J.W. Mullin: Crystallization, 3rded., Butterworth-Heinemann, Oxford 1993; A. Mersmann (ed.); Crystallization Technology Handbook, Marcel Dekker, New York 1995. Crystallization can be achieved for example by cooling, evaporation, vacuum crystallization (adiabatic cooling), reaction crystallization and salting out. The crystallization can be carried for example in stirred and unstirred tanks, in the direct-contact process, in evaporative crystallizers (R.K. Multer, Chem Eng. (N.Y.) 89 (1982) March, 87-89), in vacuum crystallizers batchwise or continuously, for example in forced-circulation crystallizers (Swenson forced-circulation crystallizer) or fluidized-bed crystallizers (Oslo type) (A.D. Randolph, M.A. Larson: Theory of Particulate Processes, 2nd ed. Academic Press, New York 1988; J. Robinson, J.E. Roberts, Can. J. Chem. Eng. 35 (1957) 105-112; J. Nyvlt Design of Crystallizers, CRC Press, Boca Raton, 1992). Fractional crystallization is also possible (L. Gordon, M.L. Salutsky, H.H. Willard: Precipitation from Homogeneous Solution, Wiley-lnterscience, New York 1959). Likewise, enantiomers and racemates can be separated (J. Jacques, A. Collet, S.H. Willen: Enantiomers, Racemates and Resolutions, Wiley, New York 1981; R.A. Sheldon: Chirotechnology, Marcel Dekker, New York 1993; A.N. Collins, G.N. Sheldrake, J. Crosby (ed.): Chirality in Industry, Wiley, New York 1985).
Conventional drying methods are described, for example in O. Krischer, W. Kast: Die wissenschaftlichen Grundlagen der Trocknungstechnik [The scientific bases of drying technology], 3rded., Springer, Berlin-Heidelberg-New York 1978; R.B. Keey: Drying: Principles and Practice, Pergamon Press, Oxford 1972; K. Kroll: Trockner und Trocknungsverfahren [Dryers and drying methods], 2nd ed., Springer, Berlin-Heidelberg-New York 1978; Williams-Gardener, A.: Industrial Drying, Houston, Gulf, 1977; K. Kroll, W. Kast: Trocknen und Trockner in der Produktion [Drying and dryers in production], Springer, Berlin-Heidelberg-New York 1989. There exist processes for convection drying, for example drying ovens, tunnel dryers, belt dryers, disk dryers, jet dryers, fluidized-bed dryers, aerated and rotating drum dryers, and spray dryers. Further processes utilize contact drying, for example blade dryers; vacuum or freeze drying; or heat radiation (infrared) or dielectric energy (microwaves) for drying.
In a preferred embodiment, the isolation of the fine chemicals from the fermentation liquor of c) is carried out by means of ion-exchange chromatography. Here, the general conditions and procedures are known to the skilled worker and described, for example in Rompp Lexikon der Chemie [Dictionary of Chemistry], 10th edition, 1997, Georg Thieme Verlag, Stuttgart; Weis, Handbuch der lonenchromatographie [Ion Chromatography Manual], 1991, VCH Verlagsgesellschaft, Weinheim. In general, a procedure will be followed in which the compound produced by the microorganisms is bound selectively on the ion exchanger and the ion exchanger is washed, for example with water, prior to elution of the compound produced by the microorganisms.

Before the solids-loaded fermentation liquor is applied to the ion-exchange chromatography column, the solids may, if appropriate, be removed by means of conventional methods with which the skilled worker is familiar, for example filtration and centrifugation.
In an especially preferred embodiment, the solids are not removed before the solids-loaded fermentation liquor is applied to the ion-exchange chromatography column. In this case, the flow of the solids-loaded fermentation liquor into the ion exchanger is advantageously against gravity so that the solids present do not lead to blocking (i.e. clogging) of the ion exchanger column.
If the metabolite produced via the microorganisms is a basic amino acid, the latter can advantageously be removed from the fermentation liquor by ion-exchange chromatography, employing an acidic cation exchanger column. In this case, the basic amino acid, for example lysine, is bound selectively on the ion exchanger column. Purification by washing, in particular with water, prior to elution is possible. Then, the basic amino acid is eluted with a suitable eluent, for example, ammonia water, preferably with 5% by volume strength ammonia water.
The use of ion-exchange chromatography for the removal or purification of basic amino acids such as lysine is described, for example, in WO 01/072689 and Lee et al., The use of ion exclusion chromatography as approved to the normal ion exchange chromatography to achieve a more efficient lysine recovery from fermentation broth, Enzyme and Microbial Technology 30 (2002), 798-303.
The fermentation liquor which remains after removal of the product of value, for example a basic amino acid such as lysine, essentially comprises the biomass produced during the fermentation, the nonmetabolized constituents of the saccharified starch solution, such as, for example, fibers and nonutilized sugars, and nonutilized buffer and nutrient salts. The secondary component can be obtained from this remaining fermentation liquor analogously to the production of bioethanol. The resulting product, which is known as distiller's dried grains with solubles (DDGS), can be sold as animal feed.
For this purpose, all of the liquor is usually partially evaporated in an evaporation step which is, as a rule, a multi-step process, and the solids present are usually decanted, for example using a decanter. The solids separated in this process have, as a rule, a dry-matter content in the range of from 10 to 80% by weight, preferably 15 to 60% by weight and especially preferably 20 to 50% by weight. Some of the liquid phase which has been removed can be returned as process water. This returned portion of the liquid phase can advantageously be employed for example in its entirety or in part in the production of the sugar-containing liquid of step a) or can be used for making buffer or nutrient salt solutions for use in the fermentation step. When admixing returned process water in step a), it must be taken into consideration that an unduly high

proportion may have an adverse effect on the fermentation owing to an unduly high presence of certain minerals and ions, for example sodium and lactate ions. Preferably, the amount of returned process water when making the suspension for the starch liquefaction is therefore limited according to the invention to a maximum of 75% by weight, preferably a maximum of 60% by weight and especially preferably a maximum of 50% by weight. Advantageously, the amount of process water when making the suspension in the preferred embodiment of step a2) is in the range of from 5 to 60% by weight and preferably from 10 to 50% by weight.
The portion of the liquid phase which is not returned into the process is concentrated in a multi-step evaporation process to give a syrup. The resulting syrup has, as a rule, a dry matter content in the range of from 20 to 90% by weight, preferably 30 to 80% by weight and especially preferably 40 to 70% by weight. This syrup is mixed with the solids removed during the decanting step and subsequently dried. Drying can be effected for example by means of a tumble dryer, a spray dryer or a paddle dryer; a tumbler dryer is preferably employed. Drying is carried out in such a way that the resulting solid has a residual moisture content of not more than 30% by weight, preferably not more than 20% by weight and especially preferably not more than 10% by weight.
If the metabolite produced by the microorganisms is methionin, the product of value is advantageously isolated by centrifugation or filtration. Here, analogous conditions and procedures as have been described for other carbon feedstocks, for example in prior application DE 10359668.2, may be used. When the fermentation has ended, fermentation liquor generated is heated to dissolve ail of the methionine. The solids are then separated off by centrifugation or filtration. The clear runoff from the solids separation step is preferably concentrated by partial or complete evaporation, during which process the methionin crystallizes out. Thereafter, the methionin is dried, if appropriate following a preceding further filtration step.
The solids separated by centrifugation or filtration essentially comprise the biomass produced during the fermentation and the nonmetabolized constituents of the saccharified starch solution, for example fibers. This remaining fermentation residue can be treated analogously to what has been described above in connection with the removal of basic amino acids, giving DDGS as secondary product.
If the metabolite produced by the microorganisms is pantothenic acid, the isolation of the product of value is likewise advantageously carried out by filtration or centrifugation. In this context, analogous conditions and procedures as have been described for other carbon feedstocks, for example in EP 1050219 and WO 01/83799, may be employed. Otherwise, work-up can be carried out analogously to what has been described above in the case of methionin. In the case of pantothenic acid, it is preferred additionally to carry out a pasteurization of all of the fermentation liquor before the solids are separated off. The clear runoff obtained from the solids separation

step is preferably partially evaporated, if appropriate treated with calcium chloride and dried, preferably spray-dried.
The remaining fermentation residue, i.e. in particular the solids which have been separated off, can be treated analogously to what has been described above in connection with the separation of basic amino acids, giving DDGS as secondary product.
If the metabolite produced by the microorganisms takes the form of polyhydroxyalkanoates, the isolation of the product of value is advantageously carried out by extraction with a solvent, such as described, for example, in US 4310684 or EP 355307. The remaining solids can be removed in the customary manner, for example by filtration or centrifugation. Otherwise, work-up can be carried out analogously to what has been described above in the case of methionin. In the case of polyhydroxyalkanoates, it is preferred additionally to carry out a pasteurization of all of the fermentation liquor before the solids are separated off. The clear runoff obtained from the solids separation step is preferably partially evaporated, if appropriate treated with calcium chloride and dried, preferably spray-dried. The further purification of the polyhydroxyalkanoates is carried out in a known manner, such as described, for example in US 4310684 or EP 355307.
The remaining fermentation residue, i.e. in particular the solids which have been separated off, can be treated analogously to what has been described above in connection with the separation of basic amino acids, giving DDGS as secondary product.
The invention furthermore relates to a process as described above, wherein
(i) a portion of not more than 50% by weight is removed from the sugar-containing liquid medium obtained in step a) which comprises the non-starchy solid constituents of the starch feedstock and a fermentation as described in b) is carried out with the remainder in order to produce a first metabolite (A); and
(it) all or some of the non-starchy solid constituents of the starch feedstock are separated from this portion and a fermentation as described in b) is carried out with this portion to produce a second metabolite (B), which is identical to or different from the metabolite (A).
In a preferred embodiment, the removal of the non-starchy solid constituents of (ii) is carried out in such a way that the solids content of the remainder of the sugar-containing liquid medium amounts to not more than 50% by weight, preferably not more than 30% by weight, especially preferably not more than 10% by weight and very especially preferably not more than 5% by weight.

This procedure makes possible, in the separate fermentation of (iii), the use of microorganisms for which certain minimum requirements, for example with regard to the oxygen transfer rate, must be met. Suitable microorganisms which are employed in the separate fermentation of (iii) are, for example, Bacillus species, preferably Bacillus subtilis. The compounds produced by such microorganisms in the separate fermentation are selected in particular from vitamins, cofactors and nutraceuticals, purine and pyrimidine bases, nucleosides and nucleotides, lipids, saturated and unsaturated fatty acids, aromatic compounds, proteins, carotenoids, specifically from vitamins, cofactors and nutraceuticals, proteins and carotenoids, and very specifically from riboflavin and calcium pantothenate.
In particular, this procedure permits the advantageous use of the process according to the invention even when the fine chemical produced is obtained, in the fermentation, as a solid.
A preferred embodiment of this procedure relates to parallel production of identical metabolites (A) and (B) in two separate fermentations. This is advantageous in particular in a case where different applications of the same metabolite have different purity requirements. Accordingly, the first metabolite (A), for example an amino acid to be used as feed additive, for example lysine, is produced using the solids-containing fermentation liquor and the same second metabolite (B), for example the same amino acid to be used as food additive, in the present case for example lysine, is produced using the solids-depleted fermentation liquor of (ii). Owing to the complete or partial removal of the non-starchy solid constituents, the complexity of the purification when working up the metabolite whose field of application has a higher purity requirement, for example as food additive, can be reduced.
In a further preferred embodiment of this procedure, the metabolite B produced by the microorganisms in the fermentation is riboflavin. To carry out the fermentation, analogous conditions and procedures as have been described for other carbon feedstocks, for example in WO 01/011052, DE 19840709, WO 98/29539, EP 1186664 and Fujioka, K.: New biotechnology for riboflavin (vitamin B2) and character of this riboflavin. Fragrance Journal (2003), 31(3), 44-48, can be employed.
For example, the following procedure may be used for carrying out this variant of the process. A preferably large-volume fermentation is implemented for the production of metabolites A, for example of fine chemicals such as lysine, in accordance with process steps a) to c) according to the invention. In accordance with (i), some of the sugar-containing liquid medium obtained in step a) is removed and freed in accordance with (ii) completely or in part from the solids by customary methods, for example centrifugation or filtration. The sugar-containing liquid medium obtained therefrom, which is essentially fully or partially freed from the solids, is, in accordance with (ii), fed to a fermentation for the production of a metabolite B, for example riboflavin. The solids stream separated in accordance with (ii) is advantageously returned to the stream of

the sugar-containing liquid medium of the large-volume fermentation.
The riboflavin-containing fermentation liquor which is thus generated in accordance with (ii) can be processed by analogous conditions and procedures as have been described for other carbon feedstocks, for example in DE 4037441, EP 464582, EP 438767 and DE 3819745. Following lyses of the cell biomass, the riboflavin, which is present in crystalline form, is separated, preferably be decanting. Other ways of separating solids, for example filtration, are also possible. Thereafter, the riboflavin is dried, preferably by means of spray dryers and fluidized-bed dryers. As an alternative, the riboflavin-containing fermentation mixture produced in accordance with (ii) can be processed under analogous conditions and using analogous procedures as described in, for example, EP 1048668 and EP 730034. After a pasteurization, the fermentation liquor is centrifuged, and the remaining solids-containing fraction is treated with a mineral acid. The riboflavin formed is removed from the aqueous-acidic medium by filtration, washed, if appropriate, and subsequently dried.
In a further preferred embodiment of this procedure, the metabolite B produced by the microorganisms in the fermentation is pantothenic acid. To carry out the fermentation, analogous conditions and procedures as have been described for other carbon feedstocks, for example in WO 01/021772, can be employed.
To carry out this process variant, a procedure such as described above for riboflavin may be followed. The sugar-containing liquid medium which has been purified in accordance with (ii) and which has preferably been essentially freed from the solids is fed into a fermentation in accordance with (ii) for the production of pantothenic acid. Here, the fact that the viscosity is reduced in comparison with the solids-containing liquid medium is particularly advantageous. The separated solids stream is preferably returned to the stream of the sugar-containing liquid medium of the large-volume fermentation.
The pantothenic-acid-containing fermentation liquor produced in accordance with (ii) can be processed under analogous conditions and using analogous procedures as have been described for other carbon feedstocks, for example in EP 1050219 and WO 01/83799. After all of the fermentation liquor has been pasteurized, the remaining solids are separated, for example by centrifugation or filtration. The clear runoff obtained in the solids separation step is partly evaporated, if appropriate treated with calcium chloride and dried, in particular spray dried.
The solids which have been separated off are processed within the scope of the large-volume fermentation process which is operated in parallel to give DDGS.
In a further preferred embodiment of this procedure, the metabolite B produced by the microorganisms in the fermentation takes the form of polyhydroxyalkanoates. To carry out the fermentation, analogous conditions and procedures as have been described for

other carbon feedstocks, for example in S.Y. Lee, Plastic Bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria, Tibtech, Vol. 14, (1996), pp. 431-438, can be employed.
To carry out this process variant, a procedure such as described above for riboflavin may be followed. The sugar-containing liquid medium which has been purified in accordance with (ii) and which has preferably been essentially freed from the solids is fed into a fermentation in accordance with (ii) for the production of polyhydroxyalkanoates. The clear runoff obtained in the solids separation step is partly evaporated, if appropriate treated with calcium chloride and dried, in particular spray dried.
The polyhydroxyalkanoate-containing fermentation liquor produced in accordance with (ii) can be processed under analogous conditions and using analogous procedures as have been described for other carbon feedstocks, for example in US 4310684 and EP 355307. After all of the fermentation liquor has been pasteurized, the remaining solids are separated, for example by centrifugation or filtration. The dear runoff obtained in the solids separation step is partly evaporated, if appropriate treated with calcium chloride and dried, in particular spray dried. The further purification of the polyhydroxyalkanoates is carried out in a manner known per se, such as, for example, as described in US 4310684 or EP 355307.
The examples which follow are intended to illustrate individual aspects of the present invention, but are in no way to be understood as limiting.
Examples
I. Milling the starch feedstock
The millbases employed hereinbelow were produced as follows. Whole maize kernels were ground completely using a rotor mill. Using different beaters, milling paths or screen elements, three different degrees of fineness were obtained. A screen analysis of the millbase by means of a laboratory vibration screen (vibration analyzer: Retsch Vibrotronic type VEl; screening time 5 minutes, amplitude: 1.5 mm) gave the results listed in Table 1.


II. Enzymatic starch liquefaction and starch saccharification
II. 1. Without phytase in the saccharification step
II. 1a) Enzymatic starch liquefaction
320 g of dry-milled corn meal (T71/03) were suspended with 480 g of water and admixed with 310 mg of calcium chloride by continuous stirring. Stirring was continued during the entire experiment. After the pH was brought to 6.5 with H2S04 and the mixture had been heated to 35°C, 2.4 g of Termamyl L were added, in the course of 40 minutes, the reaction mixture was heated to a temperature of 86.5°C, the pH being readjusted with NaOH to the above value, if necessary. Within 30 minutes, a further 400 g of the dry-milled corn meal (T71/03) were added, during which process the temperature was raised to 91 °C. The reaction mixture was held at this temperature for approximately 100 minutes. A further 2.4 g of Termamyl L were subsequently added and the temperature was held for approximately 100 minutes. The progress of the liquefaction was monitored during the experimentation using the iodine-starch reaction. The temperature was finally raised to 100°C and the reaction mixture was boiled for a further 20 minutes. At this point in time, starch was no longer detectible. The reactor was cooled to 35°C.
11.1b) Saccharification
The reaction mixture obtained in II. 1a) was heated to 61 °C, with constant stirring. Stirring was continued during the entire experiment. After the pH had been brought to 4.3 with H2So4, 10.8 g (9.15 ml) of Dextrozyme GA were added. The temperature was . held for approximately 3 hours, during which time the progress of the reaction was monitored with glucose test strips (S-Glucotest by Boehringer). The results are 'listed in Table 2 hereinbelow. The reaction mixture was subsequently heated to 80°Cand then cooled. This gave approximately 1180 g of product with a density of approximately 1.2 kg/I and a dry matter content which, as determined by infrared dryer, amounted to approximately 53.7% by weight. After washing with water, a dry matter content (without

water-soluble constituents) of approximately 14% by weight was obtained. The glucose content of the reaction mixture, as determined by HPLC, amounted to 380 g/l (see Table 2, sample No. 7).

II.2. With phytase in the saccharification step
II.2a) Starch liquefaction
A dry-milled corn meal sample is liquefied as described in II.1a).
11.2b) Saccharification
The reaction mixture obtained in II.2a) is heated to 61 °C with constant stirring. Stirring is continued during the entire experiment. After the pH has been brought to 4.3 with H2S04, 10.8 g (9.15 ml) of Dextrozyme GA and 70 μl of phytase (700 units of phytase, Natuphyt Liquid 10000L from BASF AG) is added. The temperature is held for approximately 3 hours, during which time the progress of the reaction is monitored with glucose test strips (S-Glucotest by Boehringer). The reaction mixture is subsequently heated to 80°C and then cooled. The product obtained is dried by means of infra-red dryer and washed with water. The glucose content in the reaction mixture is determined by HPLC.
IN. Construction of a lysine-overproducing C. glutamicum strain ATCC13032
lysC fdr
111.1 Construction of the plasmid pCIS lysC
In the first step of the strain construction, an allelic substitution of the wild-type gene which encodes the enzyme aspartate kinase (lysC) was carried out in C. glutamicum ATCC13032. Here, a nucleotide substitution was carried out in the lysC gene so that, in the resulting protein, the amino acid at position 311 was replaced by an lie. Starting

from the chromosomal DNA from ATCC13032 as template for a PCR reaction, lysC was amplified with the oligonucleotide primers
5'-GAGAGAGAGACGCGTCCCAGTGGCTGAGACGCATC-3( (SEQ ID NO:1) and
5'-CTCTCTCTGTCGACGAATTCAATCTTACGGCCTG»3( (SEQ ID NO:2)
with the aid of the Pfu-Turbo PCR system (Stratagene, USA) following the manufacturer's instructions. Chromosomal DNA from C. glutamicum ATCC 13032 was prepared by the method of Tauch et al. (1995) Plasmid 33:168-179 or Eikmanns et al. (1994) Microbiology 140:1817-1828. The amplified fragment is flanked at its 5' end by an Sail restriction cleavage and at its 3' end by an Mlul restriction cleavage. Prior to cloning, the amplified fragment was digested with these two restriction enzymes and purified with GFX™PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
The resulting polynucleotide was cloned via the Sail and Mlul restriction cleavages into pCLIK5 MCS integrative SacB, hereinbeiow referred to as pCIS, (SEQ ID NO: 3) and transformed into E. coli XL-1 blue. A selection for plasmid-harboring cells was achieved by plating on kanamycin (20 pg/ml) containing LB agar (Lennox, 1955, Virology, 1:190). The plasmid was isolated and the expected nucleotide sequence was verified by sequencing. The preparation of the plasmid DNA was carried out using methods and materials from Quiagen. Sequencing reactions were carried out by the method of Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74:5463-5467. The sequencing reactions were separated by means of ABI Prism 377 (PE Applied Biosystems, Weiterstadt) and evaluated. The resulting plasmid was referred to as pCIS lysC (SEQ ID NO:4). It comprises the following essential portions:


The directed mutagenesis of the C. glutamicum lysC gene was carried out using the QuickChange Kit (Stratagene, USA) following the manufacturer's instructions. The mutagenesis was carried out in the plasmid pCIS lysC (SEQ ID NO:4). The following oligonucleotide primers were synthesized for the substitution of thr 311 by 311 ile with the aid of the Quickchange method (Stratagene):
5(-CGGCACCACCGACATCATCTTCACCTGCCCTCGTTCCG -3' (SEQ ID NO:5)
5(-CGGAACGAGGGCAGGTGAAGATGATGTCGGTGGTGCCG -3' (SEQ ID NO:6)
The use of these oligonucleotide primers in the Quickchange reaction leads, in the lysC gene (SEQ ID NO:7), to a substitution of the nucleotide in position 932 (of C by T). The resulting amino acid substitution Thr311lle in the lysC gene is verified by the sequencing reaction after transformation into E. coli XL1-blue and plasmid preparation. The plasmid was named pCIS lysC thr311ile (SEQ ID NO:8). It comprises the following essential portions:

III.3 Transformation of pCIS lysC thr311ile into C. glutamicum (strain ATCC13032)
The plasmid pCIS lysC thr311ile was transformed into C. glutamicum ATCC13032 by means of electroporation as described by Liebl et al., FEMS Microbiology Letters 53:299-303 (1989). Modifications of the protocol are described in DE 10046870. The chromosomal arrangement of the lysC locus of individual transformants was verified using standard methods by means of Southern blot and hybridization as described in Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor (1989). It was thereby ensured that the transformants were those which have the transformed plasmid integrated at the lysC locus by homologous recombination. After such colonies have been grown overnight in media without antibiotic, the cells are plated onto a sucrose CM agar medium (10% sucrose) and incubated for 24 hours at 30°C.
Since the sacB gene which is present in the vector pCIS lysC thr311 the converts sucrose into a toxic product, only those colonies which have the sacB gene deleted by

a second homologous recombination step between the wild-type gene lysC and the mutated gene lysC thr311ile are capable of growing. During the homologous recombination, either the wild-type gene or the mutated gene can be deleted together with the sacB gene. When the sacB gene is removed together with the wild-type gene, a mutated transformant results.
Growing colonies were picked out and studied for a kanamycin-sensitive phenotype. Clones with deleted sacB must simultaneously demonstrate kanamycin-sensitive growth behavior. Such kanamycin-sensitive clones were studied for their lysine productivity in a shake flask. For comparison, the untreated C. glutamicum ATCC13032 was grown. Clones whose lysine production was increased over the control were selected, chromsomal DNA was obtained, and the corresponding region of the lysC gene was amplified by a PCR reaction (Pfu-Turbo PCR Systems; Stratagene, USA) following the manufacturer's instructions and sequenced (by the method of Sanger et al., loc. cit.). Such a clone with the characteristic of enhanced lysine synthesis and confirmed mutation in lysC at position 932 was referred to as ATCC13032 iysCfbr.
Example 1
a) Enzymatic starch liquefaction and starch saccharification
500 g of dry-milled corn meal were suspended in 750 ml of water and again milled finely in a blender. The suspension was divided into 4 samples No. 1 to No. 4, each of which was treated with approximately 3 g of heat-stable a-amylase (samples No. 1 and 2: Termamyl L; samples No. 3 and 4: Spezyme). Samples No. 2 and 4 were subsequently treated with approx. 7 g/l glucoamylase (sample No. 2: Dextrozyme GA; sample no. 4: Optidex). This gave pale yellow viscous samples whose solids content was in each case separated by centrifugation, during which process a layer of hydrophobic solids floated on top of the clear liquid phase.
The clear supernatant of the resulting samples, in concentrated form and in 10-fold dilution, was analyzed by means of HPLC either ignoring or taking into consideration the spun-down pellet. When the pellet was taken into consideration, a pellet dry-matter content of 50% by weight was assumed. The results, based on the starting sample, are listed in Table 3 hereinbelow.


b) Fermentation
Two maize meal hyclrolyzates obtained in accordance with Example 11.1 were employed in shake-flask experiments using Corynebacterium glutamicum (flasks 4-9). In addition, a wheat flour hydrolyzate prepared analogously to Example 11.1 was used in parallel (flasks 1-3).
1b.1) Preparation of the inoculum
The cells are streaked onto sterile CM agar (composition: see Table 4; 20 minutes at 121°C) and then incubated for 48 hours at 30°C. The cells are subsequently scraped from the plates and resuspended in saline. 25 ml of the medium (see Table 5) in 250 ml Erlenmeyer flasks are inoculated in each case with such an amount of the cell suspension thus prepared that the optical density reaches an OD600 value of 1 at 600 nm.


1 b.2) Preparation of the fermentation liquor
The compositions of the flask media 1 to 9 are listed in Table 5.

After the inoculation, the flasks were incubated for 48 hours at 30°C and with shaking (200 rpm) in a humidified shaker. After the fermentation was terminated, the sugar and lysine contents were determined by HPLC. The HPLC was carried out with an Agilent 1100 series LC system. Pre-column derivatization with ortho-phthaldehyde permits the quantification of the amino acid formed; the product mixture is separated using an Agilent Hypersil AA column. The results are compiled in Table 6.


In all flasks, lysine was produced in comparable amounts in the order of approximately 30 to 40 g/l, corresponding to the yield obtained in a standard fermentation with glucose nutrient solution.
Example 2: Fermentation
Using a cornmeal hydrolyzate obtained as described in Example 11.1, a fermentation is carried out analogously to Example 1b), using the strain ATCC13032 lysCfer described under III. The cells are incubated on sterile CM agar (composition Table 4; 20 minutes at 121°C) for 48 hours at 30°C. The cells are subsequently scraped from the plates and resuspended in saline. 25 ml of the medium 1 or 2 (see Table 5) in 250 ml Erlenmeyer flasks are inoculated in each case with such an amount of the cell suspension thus prepared that the optical density reaches an OD500 value of 1 at 610 nm. The samples are then incubated for 48 hours at 200 rpm and 30°C in a humidified shaker (relative atmospheric humidity 85%). The lysine concentration in the media is determined by HPLC. In all cases, approximately identical lysine quantities were produced.We claim:
1. A process for the production of at least one microbial metabolite having at least 3 carbon atoms, or having at least 2 carbon atoms and at least 1 nitrogen atom by means of sugar-based microbial fermentation, comprising:
a) the preparation of a sugar-containing liquid medium from a starch feedstock, the sugar-containing liquid medium having a monosaccharide content of more than 20% by weight and also comprising non-starchy solid constituents of the starch feedstock;
b) the fermentation for the production of the metabolite(s) using the sugar-containing liquid medium; and
c) depletion or isolation of at least one metabolite from the fermentation liquor,
which comprises culturing, by using the sugar-containing liquid medium, a microorganism strain which produces the desired metabolite(s), which liquid medium is obtained by:
a1) milling the starch feedstock; and
a2) liquefying the millbase in an aqueous liquid in the presence of at least one starch-liquefying enzyme, followed by saccharification using at least one saccharifying enzyme, where at least some of the millbase is added continuously or batchwise to the aqueous liquid in the course of the liquefaction step
2. The process according to claim 1, wherein the sugar-containing liquid medium comprises at least 20% by weight of the total non-starchy solid constituents of the starch source.
3. The process according to claim 1 or 2, wherein the at least one starch-iiquefyihg enzyme is selected from a-amyiases and the at least one saccharifying enzyme from glycoamylases.
4. The process according to one of the preceding claims, wherein cereal kernels are used as starch feedstock.
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5. The process according to claim 4, wherein the cereal is selected from corn, rye, triticai and wheat kernels.
6. The process according to one of the preceding claims, wherein the millbase obtained during milling in step a1) comprises at least 50% by weight of flour particles with a particle size of more than 100 μm.
7. The process according to one of the preceding claims, wherein the liquefaction and saccharifying of the millbase in step a2) is carried out in such a way that the viscosity of the liquid medium amounts to not more than 20 Pas.
8. The process according to one of the preceding claims, wherein at least 25% by weight of the total amount of the millbase added during the liquefaction are added at a temperature above the gelling temperature of the starch present in the millbase.
9. The process according to one of claims 3 to 8, wherein, in step a2), some of the at least one oamyiase is added to the aqueous liquid during the liquefaction.
10. The process according to one of the preceding claims, wherein a sugar-containing liquid medium with a monosaccharide content of more than 40% by weight is obtained.
11. The process according to one of the preceding claims, wherein at least one phytase is added to the sugar-containing liquid medium before the fermentation step b).
12. The process according to one of the preceding claims, wherein the metabolites produced are selected from non-volatile substances.
13. The process according to one of the preceding claims, wherein the metabolites produced are selected from organic mono-, di- and tricarboxylic acids which optionally have hydroxyl groups attached to them and which have 3 to 10 carbon atoms, among proteinogenic and nonproteinogenic amino acids, purine bases, pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols having 3 to 10 carbon atoms, higher-functionality alcohols having 3 or more hydroxyl groups, longer-chain alcohols having at least 4 carbon
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atoms, carbohydrates, aromatic compounds, vitamins, provitamins, cofactors, nutraceuticals, proteins, carotenoids, ketones having 3 to 10 carbon atoms, lactones, biopolymers and cyclodextrins.
14. The process according to one of the preceding claims, wherein the metabolites produced are selected from enzymes, amino acids, vitamins, disaccharides, aliphatic mono- and dicarboxylic acids having 3 to 10 carbon atoms, aliphatic hydroxycarboxylic acids having 3 to 10 carbon atoms, ketones having 3 to 10 carbon atoms, alkanols having 4 to 10 carbon atoms, alkanediols having 3 to 8 carbon atoms and polyhydroxyalkanoates.
15. The process according to one of the preceding claims, wherein the microorganisms are selected from natural or recombinant microorganisms which produce at least one of the following metabolites: enzymes, amino acids, vitamins, disaccharides, aliphatic mono- and dicarboxylic acids having 3 to 10 carbon atoms, aliphatic hydroxycarboxylic acids having 3 to 10 carbon atoms, ketones having 3 to 10 carbon atoms, alkanols having 4 to 10 carbon atoms, alkanediols having 3 to 8 carbon atoms and polyhydroxyalkanoates.

6. The process according to claim 15, wherein the microorganisms are selected from the genera Corynebacterium, Bacillus, Ashbya, Escherichia, Aspergillus, Alcaligenes, Actinobacillus, Anaerobiospirillum, Lactobacillus, Propionibacterium and Clostridium, in particular among strains of Corynebacterium glutamicum, Bacillus subtilis, Ashbya gossypii, Escherichia coli, Aspergillus niger or Alcaligenes latus, Anaerobiospirillum succiniproducens, Actinobacillus succinogenes, Lactobacillus delbruckii, Lactobacillus leichmanni, Propionibacterium arabinosum, Propionibacterium schermanii, Propionibacterium freudenreichii, Clostridium propionicum and Clostridium acetobutlicum.
7. The process according to one of the preceding claims, wherein the depletion or isolation of the metabolites from the fermentation liquor as described in step c) is carried out by means of ion-exchange chromatography.
8. The process according to claim 17, wherein the metabolite is bound selectively on the ion exchanger and, if appropriate, the ion exchanger is -washed prior to elution of the product.
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19. The process according to one of claims 17 or 18, wherein the solids-loaded fermentation liquor flows towards the ion exchanger against gravity.
20. The process according to one of the preceding claims, wherein
(i) a portion of not more than 50% by weight is removed from the sugar-containing liquid medium obtained in step a) which comprises the non-starchy solid constituents of the starch feedstock and a fermentation as described in b) is carried out with the remainder in order to produce a first metabolite (A); and
(ji) all or some of the non-starchy solid constituents of the starch feedstock are separated from this portion and a fermentation as described in b) is carried out with this portion to produce a second metabolite (B), which is identical to or different from the metabolite (A).
21. The process according to claim 20, wherein the separation of the non-starchy solid constituents of (ii) is carried out in such a way that the solids content of the remainder of the sugar-containing liquid amounts to not more than 50% by weight.
22. The process according to claim 20 or 21, wherein the metabolite (B) is selected from phytase, riboflavin, pantothenic acid and polyhydroxyalkanoates.
23. The process according to one of the preceding claims, wherein, after the depletion or isolation of the metabolite in accordance with step c), the volatile constituents of the fermentation liquor are removed to at least some extent, giving a solid or semisolid protein composition.
24. A protein composition, obtainable by a process according to claim 23.
25. The protein composition according to claim 24, essentially comprising the following dry matter constituents:

a) 1 to 90% by weight of biomass from the fermentation;
b) 1 to 90% by weight of non-starchy constituents of the starch source;
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c) 0.01 to 10% by weight of a microbial metabolite having at least 3 carbon
atoms or at least 2 carbon atoms and at least one nitrogen atom;
d) 0 to 90% by weight of customary formulation auxiliaries; and
e) 0 to 40% by weight of unmetaboiized further constituents of the fermentation liquor;
where the components a) to e) add up to 100% by weight of dry matter.
16. The protein composition according to claim 24 or 25 with a crude protein content in the range of from 40 to 90% by weight, based on the dry matter of the protein composition.
11. The protein composition according to one of claims 24 to 26 which features at least one essential amino acid from among lysine, methionine, threonine and tryptophan.
28. The use of a sugar-containing liquid medium as defined in any of claims 1 to 22 for the fermentative production of a microbial metabolite with at least 3 carbon atoms or with at least 2 carbon atoms and at least one 1 nitrogen atom.

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